Characterization, Expression Profiling, and Biochemical Analyses of the Cinnamoyl-CoA Reductase Gene Family for Lignin Synthesis in Alfalfa Plants

Cinnamoyl-CoA reductase (CCR) is a pivotal enzyme in plant lignin synthesis, which has a role in plant secondary cell wall development and environmental stress defense. Alfalfa is a predominant legume forage with excellent quality, but the lignin content negatively affects fodder digestibility. Currently, there is limited information on CCR characteristics, gene expression, and its role in lignin metabolism in alfalfa. In this study, we identified 30 members in the CCR gene family of Medicago sativa. In addition, gene structure, conserved motif, and evolution analysis suggested MsCCR1–7 presumably functioned as CCR, while the 23 MsCCR-likes fell into three categories. The expression patterns of MsCCRs/MsCCR-likes suggested their role in plant development, response to environmental stresses, and phytohormone treatment. These results were consistent with the cis-elements in their promoters. Histochemical staining showed that lignin accumulation gradually deepened with the development, which was consistent with gene expression results. Furthermore, recombinant MsCCR1 and MsCCR-like1 were purified and the kinetic parameters were tested under four substrates. In addition, three-dimensional structure models of MsCCR1 and MsCCR-like1 proteins showed the difference in the substrate-binding motif H212(X)2K215R263. These results will be useful for further application for legume forage quality modification and biofuels industry engineering in the future.


Introduction
Alfalfa (Medicago sativa L.) is a worldwide-cultivated legume forage, with high nutrition and good palatability [1]. Digestibility is part of the key indicators of forage quality, which has a significant influence on animal performance. Generally, forage digestibility is affected by various cellular components, including lignin content [2]. Genetically reducing lignin content in forage legumes can improve digestibility and animal performance, as well as commercialization for forage quality improvement [3]. For example, the caffeic acid O-methyltransferase downregulated transgenic tall fescue plants showed reduced lignin content and significantly increased digestibility [4]. When fed adult horses with reduced lignin alfalfa hay, an improvement in the dry matter digestibility was found, with no change in forage consumption, fecal particle size, or digesta retention time [5]. By using transgenic alfalfa lines with downregulated cytochrome P450 enzymes in lignin pathway, it showed a strong negative relationship between lignin content and rumen digestibility but not between lignin composition and digestibility [6]. However, it is difficult to draw a conclusion on whether the changes in lignin monomer could benefit biomass digestibility because there is a lack of unified research background in the previous studies [7]. In addition, lignin also has a negative impact on the conversion of lignocellulose biomass into cellulosic ethanol in the biofuels production industry [8][9][10]. Interestingly, alfalfa is considered a potential feedstock for biofuels due to its being valuable in lignocellulosic biomass [11]. Thus, lignin content modification is an important strategy for both forage and biofuel engineering, with no effect on the plant yield [12].
Lignin is a group of polyphenolic polymers that are deposited predominantly in the thickened secondary cell walls [13]. The evolution of lignin was accompanied by the emergence of the vascular land plants and exhibited both distinct subcellular localization and monomeric composition in specific cell types [14]. Plants with loss-of-function of genes in lignin biosynthesis, such as cinnamoyl-CoA reductase (CCR), generally showed collapsed xylem and/or dwarf phenotype [15]. Moreover, lignification is one of the responses of plant cells to various environmental stresses. There is a close relationship between lignin accumulation and environmental stresses in many plant species. For example, in different Iranian cultivars of basil, water deficit stress leads to increasing expression of genes in lignin synthesis [16]. Under copper (Cu 2+ ) stress condition, Panax ginseng suspension cultures showed an increased accumulation of phenolics and lignin, which reflects the protective response to Cu 2+ -induced cell damage [17]. During the plant-environment interaction, the functional integrity of the plant cell needs the maintenance of cell wall integrity. Stresses-induced cell wall damage could initiate the secondary reactive oxygen species (ROS) burst and jasmonic acid (JA) accumulation, followed by a negative feedback loop that represses each other's production and subsequent lignin accumulation [18]. In addition, lignin content and the expression of lignin-metabolism-related genes were found to play critical roles in lodging resistance of common buckwheat and barley [19,20]. Interestingly, genetically modified plants with decreased lignin biosynthesis can indirectly influence the synthesis of other secondary metabolites and the expression of stress-related genes [21].
Lignin is synthesized in the spaces between the cell wall polysaccharides by the oxidative coupling of three monolignols, coniferyl alcohol, sinapyl alcohol, and p-coumaryl alcohol, and then there is incorporation into lignin to form guaiacyl (G), syringyl (S), and p-hydroxyphenyl (H) subunits, respectively [14,22]. In plants, lignin monomers are synthesized from phenylalanine in the cytoplasm by two of a total of three branches. The first branch, which is called the general phenylpropanoid pathway, is from phenylalanine to p-coumaroyl-CoA and subsequent feruloyl-CoA. This pathway is shared with some important secondary metabolites, such as flavonoids and coumarins [23,24]. The second branch is called monolignol pathway, in which hydroxycinnamoyl-CoA esters are reduced by CCR (EC 1.2.1.44) and cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) to generate monolignols [25]. CCR catalyzes the p-coumaryl-CoA, caffeoyl-CoA, and feruloyl-CoA to form p-coumaraldehyde, caffeyl aldehyde, and coniferaldehyde, respectively, and is further converted to the corresponding monolignols, coumaryl alcohol, caffeoyl alcohol, and feruloyl alcohol, with the CAD catalyst [14]. Common to all three branches of the lignin synthesis pathway, genetic changes of CCR and CAD genes tend to exhibit overall reduced lignin content but not one or two monomer(s) [7,26,27]. Therefore, CCR is a pivotal enzyme involved in monolignols biosynthesis in plants.
The CCR gene family exhibits diversity in different plant species. Two CCR genes and five CCR-like genes were found in Arabidopsis thaliana. AtCCR1 plays a role in lignification during the plant developmental stage, and AtCCR2 is functionally expressed in stress and pathogen response [28,29]. The mutant of AtCCR1 exhibits decreased lignin content, while triple mutant with the loss-function of CAD-C, CAD-D, and CCR1 contains 50% of wild-type lignin content and shows a severe dwarf phenotype and male sterility [30]. Reintroduced CCR1 expression specifically in the protoxylem and metaxylem vessel cells in the mutant of AtCCR1 can effectively overcome the vascular collapse and stem biomass yield decrease, regardless of the similar cell wall composition and metabolome compared with AtCCR1 mutation plants [31]. In switchgrass, PvCCR1 and PvCCR2 were found to possess CCR activity, whereas PvCCR1 might function in lignin biosynthesis during development stage and PvCCR2 might be involved in stress defense [32]. In Populus trichocarpa, Shi et al. [33] identified 11 CCRs, and PtrCCR2 showed high expression in differentiating xylem and significantly less in other tissues. In model legume Medicago truncatula, Zhou et al. [34] have identified two CCR genes, in which MtCCR1 had a critical effect on plant growth, while MtCCR2 exhibited moderate reduced lignin content and little influence on growth phenotype. In rice, the OsCCR knockdown plants showed a decrease in lignin deposition in root and anther. Interestingly, a component of an SCF E3 ligase OsFBK1 mediated OsCCR degradation, suggesting the role of OsFBK1 and OsCCR in the development of rice anthers and roots [35]. However, there is still little knowledge of the characteristics of the CCR family genes that respond to lignin biosynthesis in widely cultivated forage M. sativa.
Cultivated alfalfa is an autotetraploid (2n = 4x = 32), so it generates a lot of trouble in the identification of its genes and molecular genetic research. Recently, alfalfa (M. sativa L.) genome sequencing has been completed, in which it was reported with 3010 Mb of genome size [36]. This provides a great opportunity for the genome-wide analysis of the CCR gene family. In this study, we searched the alfalfa genome database to identify CCR family members and further analyzed the gene structures, evolutionary relationships, and gene expression patterns. Finally, we cloned and further recombinantly expressed MsCCR1 and MsCCR-like1, followed by the kinetic parameters and three-dimensional structure analysis, to explore the MsCCRs protein structure and possible function in lignin biosynthesis.

Identification and Analysis of Cinnamoyl-CoA Reductase (CCR) Genes in M. sativa Genome
In total, 30 putative CCR gene sequences were identified from M. sativa genome database by domain confirmation and homology research with the signature motif KN-WYCYGK. Among them, 7 CCR genes were identified with full-length CCR motif sequences, and 23 sequences with partial CCR motifs were characterized as CCR-like genes (Table S1). Of the 23 CCR-like genes, 7 CCR-like genes (namely MsCCR-like1-7) are homologous to M. truncatula CCR-like genes SNL6-1/2, and the other 16 CCR-like genes (namely MsCCR-like8-23) are homologous to MtCCR1-3/4. The molecular weight of 30 predicted MsCCR/MsCCR-like proteins varied from 32.35 to 39.85 kDa, with isoelectric points (pI) in the range of 5.00-7.48.

Gene Structure, Evolution, and Conserved Motif Analysis of MsCCRs/MsCCR-like Genes
To find out evolutionary relationships among members of the CCRs from alfalfa, a neighbor-joining (N-J) tree was constructed based on the nucleotide sequences ( Figure 1A). The 30 MsCCRs/MsCCR-likes sequences were divided into 5 subfamilies, and subfamily I contained all 7 MsCCR genes, while MsCCR-like genes were classified in subfamilies II-V ( Figure 1A). Furthermore, the intron/exon structure of MsCCRs/MsCCR-like genes was shown ( Figure 1B). It can be clearly seen that genes in the same subfamily have similar gene structures. In turn, there are distinct differences in gene structures among the different subfamilies, with the different numbers of introns from 2-5. For example, subfamily IV has 2 introns, subfamily V has 5 introns, and most of the CCR genes (subfamilies I, II, and III) contain 4 introns. By using the MEME online software, 20 conserved motifs in the MsCCRs were captured, and further analyses were carried out ( Figure 1C). Furthermore, the sequence of each motif was analyzed ( Table S2). All of the MsCCR/MsCCR-like proteins contain motifs 1-6. Proteins in groups I, IV, and V contain motifs 7 and 9, while proteins in groups I and V contain motif 8. Furthermore, proteins in groups I and V mainly lack the motifs 11-13, 15, 19, and 20. In addition, motifs 18, 15, 20, and 16 are unique to proteins in groups I, III, IV, and V, respectively. We further constructed two phylogenetic trees with CCRs in alfalfa and other plants to clarify their relationships and predict the possible biological functions ( Figure 2). Three CCR-like genes and 19 CCR genes from different plants, including M. truncatula, Arabidopsis thaliana, Oryza sativa, Zea mays, Triticum aestivum, Solanum tuberosum, Trifolium pretense, Leucaena leucocephala, Eucalyptus gunnii, Pinus radiate, Picea abies, and Populus trichocarpa, were selected from NCBI [37] (Table S3). All CCRs/CCR-like proteins fell into two groups: the CCR bona fide clade and the CCR-like clade. For example, all the 23 MsCCR-like proteins and one AtCCR-like protein as well as two MtCCR-like SNLs were clustered into the CCR-like clade, and the others were parceled into the CCR bona fide clade. The CCR bona fide clade was further classified into three subfamilies: dicot clade, monocot clade, and gymnosperm clades, and 7 MsCCRs were clustered together with LlCCR1, EgCCR, AtCCR1/2, PtCCR, PtrCCR, and PbCCR1 ( Figure 2A). Furthermore, many identified CCRs with the function related to lignin biosynthesis exhibit a close relationship to MsCCRs (in Group I) in the radiation tree ( Figure 2B). These results indicated possible functional diversity between CCR and CCR-like proteins.
Alignments among MsCCR/MsCCR-like proteins revealed that all of them have the NAD(P)-binding motif GXXGXXA/G. MsCCRs and MsCCR-like12-22 also have the NADP + specific motif R(X) 5 K, which was absent in other MsCCR-likes ( Figure 3). In addition, only MsCCRs possess the motif H212(X) 2 K215R263 residuce, which was found to be critical for substrate binding in PhCCR1 [38]. Interestingly, these were in agreement with the results in the CCR signature motif KNWYCYGK. The diversity of conserved motifs in MsCCR-like proteins indicates that they might have different activities and biological functions.

The Cis-Acting Regulatory Elements in the Promoter of MsCCRs
The regulation of gene expression in organ cells is a complex process, and transcriptional activation of promoters is a vital event. The promoter of MsCCR/MsCCR-like

The Cis-Acting Regulatory Elements in the Promoter of MsCCRs
The regulation of gene expression in organ cells is a complex process, and transcriptional activation of promoters is a vital event. The promoter of MsCCR/MsCCR-like genes contained a variety of cis-acting elements, including ABRE, ARE, BOX4, G-Box, GT1-motif, MYB, and MYC ( Figure 4). For example, the promoters of MsCCR1-4 have light-related cis-acting elements (GT1-motif, Box4, and G-Box), ARE, as well as MYB-and MYC-binding elements, suggesting the expression of MsCCR1-4 might be involved in light stimulus, anaerobic induction, stress response, and plant development [39]. The

The Expression Patterns of MsCCR/MsCCR-like Genes and Lignin Content Analysis d Different Growth Stage
RT-qPCR was then performed to examine the role of MsCCR/MsCCR-like gen alfalfa during different developmental stages. According to the specificity of CD quences, 6 pairs of specific primers were obtained, for example, Group Ia (MsCCR Group Ib (MsCCR5-7), Group IV (MsCCR-like8-11, 23), Group III (MsCCR-like5-7), G II (MsCCR-like1-4), and MsCCR-like17. As shown in Figure 5A, the transcrip MsCCR1-4 (Group Ia) were upregulated, especially in the shoot after 28 days of gr Meanwhile, the transcripts of MsCCR5-7 (Group Ib) did not change considerably, higher levels underground than aboveground. Unexpectedly, transcripts of gen Group IV showed more abundance than Group Ia, and increased expression wit development process was also shown. In addition, the expression pattern of gen Group III was similar to Group Ib, and Group II was similar to Group Ia. The transcr MsCCR-like17 exhibited slight induction, especially in leaves. Meanwhile, histoche staining was employed to explore the lignin levels in the alfalfa plant stem and roo coloration gradually deepened with the development process, especially after 28 da growth.

The Expression Patterns of MsCCR/MsCCR-like Genes and Lignin Content Analysis during Different Growth Stage
RT-qPCR was then performed to examine the role of MsCCR/MsCCR-like genes in alfalfa during different developmental stages. According to the specificity of CDS sequences, 6 pairs of specific primers were obtained, for example, Group Ia (MsCCR1-4), Group Ib (MsCCR5-7), Group IV (MsCCR-like8-11, 23), Group III (MsCCR-like5-7), Group II (MsCCR-like1-4), and MsCCR-like17. As shown in Figure 5A, the transcripts of MsCCR1-4 (Group Ia) were upregulated, especially in the shoot after 28 days of growth. Meanwhile, the transcripts of MsCCR5-7 (Group Ib) did not change considerably, with higher levels underground than aboveground. Unexpectedly, transcripts of genes in Group IV showed more abundance than Group Ia, and increased expression with the development process was also shown. In addition, the expression pattern of genes in Group III was similar to Group Ib, and Group II was similar to Group Ia. The transcript of MsCCR-like17 exhibited slight induction, especially in leaves. Meanwhile, histochemical staining was employed to explore the lignin levels in the alfalfa plant stem and root. The coloration gradually deepened with the development process, especially after 28 days of growth.

The Expression Patterns of MsCCRs/MsCCR-likes under Abiotic Stresses and Hormone Treatment
Further experiments were performed to detect the response of MsCCR/MsCCR-like genes under various stresses and hormone treatments. As shown in Figure 6A, with cold (4 °C), NaCl, PEG6000, AlCl3, and CdCl2 treatment, the transcript levels of genes in Group Ia and Group II were slightly increased at early (1 h) and late stage (48 h), but downregulated (especially under Al stress) at 6 and 24 h of treatment. Meanwhile, the transcripts of genes in Group Ib and Group III showed a similar trend but it was more obvious under 4 °C and NaCl treatments. The genes in Group IV were mainly induced at 3-24 h with 4 °C and NaCl treatments, and there was strong induction at 1-3 h and reduction at 48 h. The transcript of MsCCR-like17 showed decreased expression after 3-24 h of 4 °C, NaCl, PEG6000, AlCl3, and CdCl2 treatments. After 1 h of ultraviolet-B (UV-B) treatment and subsequent recovery period, genes in Group Ia/Ib/II/III were upregulated, while MsCCR-like17 and genes in Group IV showed a downregulated trend during stress treatment and recovery periods.

The Expression Patterns of MsCCRs/MsCCR-likes under Abiotic Stresses and Hormone Treatment
Further experiments were performed to detect the response of MsCCR/MsCCR-like genes under various stresses and hormone treatments. As shown in Figure 6A, with cold (4 • C), NaCl, PEG6000, AlCl 3 , and CdCl 2 treatment, the transcript levels of genes in Group Ia and Group II were slightly increased at early (1 h) and late stage (48 h), but downregulated (especially under Al stress) at 6 and 24 h of treatment. Meanwhile, the transcripts of genes in Group Ib and Group III showed a similar trend but it was more obvious under 4 • C and NaCl treatments. The genes in Group IV were mainly induced at 3-24 h with 4 • C and NaCl treatments, and there was strong induction at 1-3 h and reduction at 48 h. The transcript of MsCCR-like17 showed decreased expression after 3-24 h of 4 • C, NaCl, PEG6000, AlCl 3 , and CdCl 2 treatments. After 1 h of ultraviolet-B (UV-B) treatment and subsequent recovery period, genes in Group Ia/Ib/II/III were upregulated, while MsCCR-like17 and genes in Group IV showed a downregulated trend during stress treatment and recovery periods.
After exogenous salicylic acid (SA) treatment, the transcripts of genes in Group Ia/Ib/III were slightly upregulated at 6 h but decreased after 24 h of treatment ( Figure 6B After exogenous salicylic acid (SA) treatment, the transcripts of genes in Group Ia/Ib/III were slightly upregulated at 6 h but decreased after 24 h of treatment ( Figure 6B). The abscisic acid (ABA), nitric oxide donor sodium nitroprusside (SNP), and ethephon (ETH) treatments showed similar trends with the upregulated expression of genes in Group Ia/Ib/II at the late stage (48 h) of treatment. The genes in Group III showed a downregulated trend before 24 h of ABA, SNP, and ETH treatment but were upregulated after 48 h of treatments. In addition, the transcripts in Group IV were increased during the first 24 h but decreased after 48 h of SA, ABA, and SNP treatments, and they showed , and ETH (500 µM) treatment, the transcripts were analyzed by RT-qPCR. UV-B (1 J·m −2 ·h −1 ) was treated for 6 h, followed by 12 h of recovery (in grey). Data were from three independent experiments with at least three replicates for each. Actin2 was used as internal reference, and the relative expression of each gene was further compared to control samples at the same time point under each treatment.

Cloning, Heterologous Expression, and Enzymatic Assay of MsCCR1 and MsCCR-like1 Gene
To illustrate the biochemical functions of MsCCRs, we cloned MsCCR1 (in Group I) and MsCCR-like1 (in Group II) gene. Heterologous expression of the His-tagged MsCCR1 and MsCCR-like1 was successfully obtained from E. coli by 0.2 mM isopropyl-β-D -thiogalactopyranoside (IPTG) induction at 16 • C. Recombinant MsCCRs were purified with Ni 2+ affinity chromatography, followed by molecular masses analysis through SDS-PAGE. The molecular masses of the MsCCR1 exhibited about 37.21 kDa, and MsCCR-like1 was about 33.61 kDa, which is consistent with their theoretical molecular mass ( Figure S2).
Furthermore, the kinetic parameters of recombinant MsCCR1 and MsCCR-like1 to different substrates, including feruloyl-, p-coumaroyl-, caffeoyl-, and sinapoyl-CoAs, were investigated, respectively. As shown in Table 1, MsCCR1 catalyzed feruloyl-CoA, pcoumaroyl-CoA, and sinapoyl-CoA, but it had the lowest Michaelis constant value (K m ) for sinapoyl-CoA. In addition, the catalytic efficiency (K cat /K m ) of MsCCR1 for sinapoyl-CoA was more than twice that for feruloyl-CoA and p-coumaroyl-CoA. Correspondingly, MsCCR-like1 catalyzed p-coumaroyl-CoA and caffeoyl-CoA, with similar K m and K cat /K m . The different characters in catalyzing suggest their diverse functions in lignin synthesis.

Three-Dimensional Structure Analysis of CCRs in M. sativa and Other Plants
The three-dimensional structure models of MsCCR1 and MsCCR-like1 proteins in the bona fide clade were constructed ( Figure 7A,B). It can be seen that a part of the MsCCR-like1 in the C terminal is missing, which including the substrate-binding motifs H212(X)2K215R263 ( Figure 7C,D). Furthermore, three-dimensional structure models of some representative members MsCCR5, MsCCR-like7/11/17/21, and typical CCR protein in other species, including MtCCR, AtCCR1/2, TaCCR2, EgCCR, PtCCR, LlCCR1, ZmCCR2, and PaCCR2, were also constructed ( Figure S3). The structures of MsCCR1, MsCCR5, and the identified CCRs in other plants share high similarities, but MsCCR-likes exhibited structural changes within them in some parts of the protein ( Figure S4). In addition, the alignment of the above CCR/CCR-like proteins showed that some key amino acid residues are missing in MsCCR-likes compared to CCRs. These results suggest that the catalytic function of the MsCCR-likes might be different from that of MsCCRs.

Discussion
CCR plays a central role in lignin biosynthesis, and it is considered to be a biotechnological target in lignocellulosic biomass engineering [40]. Meanwhile, it is also the target to decrease lignin content in alfalfa because of its enormous influence on forage digestibility [41]. However, little is known about the CCR gene family in the widely cultivated forage M. sativa. In this study, we identified 7 MsCCR genes with the location at

Discussion
CCR plays a central role in lignin biosynthesis, and it is considered to be a biotechnological target in lignocellulosic biomass engineering [40]. Meanwhile, it is also the target to decrease lignin content in alfalfa because of its enormous influence on forage digestibility [41]. However, little is known about the CCR gene family in the widely cultivated forage M. sativa. In this study, we identified 7 MsCCR genes with the location at chromosomes 2.1-2.4 and 4.1-4.3 in M. sativa (Figures 1 and S1; Table S1). The proteins encoded by MsCCR1-4 (with the same protein length and pI) and MsCCR5-7 (with the same protein length and pI) are homologous to functionally characterized MtCCR1 and MtCCR1-2, respectively (Table S1; [34,42]). The deficiency of the CCR gene in chromosome 4.4 suggested chromosomal fragment loss events during evolution. In addition, we also identified 23 MsCCR-likes with partially altered motif KNWYCYGK. The phylogenetic trees showed they are clustered together with a closed relationship to AtCCR-like and MtCCR-likes (Figure 2).
Diverse CCR gene families have been reported in some plant species. In Arabidopsis, 11 putative CCR genes have been identified [43]. In addition, there are 11 CCRs in Populus tomentosa, 33 in Oryza sativa, 10 in Eucalyptus grandis, 31 in pear (Pyrus bretschneideri Rehd.), and 9 in Poplus trichocarpa [7,24,33,44,45]. The CCR member in most diploid plants is almost half in the tetraploid plant such as alfalfa and some diploid plants with wholegenome duplication events such as Chinese white pear. In addition, this is also the reason that there have been many tandem duplication events during alfalfa evolution, which can be concluded from the synteny analysis of CCR/CCR-like genes in M. sativa and M. truncatula ( Figure S1).
The cis-element predicted in the promoter of MsCCR/MsCCR-like genes suggested the involvement of MsCCR/MsCCR-like in plant development (MYB/MYC), phytohormone response (ABRE), and stresses response, such as light (GT1-motif), anaerobic induction (ARE), and MYB-involved abiotic stress responses (Figure 4; [46,47]). Further expression profiling showed that MsCCR1-4 (Group Ia) and MsCCR-like1-4, 8-11, 23 (Groups II and IV) are mainly involved in lignin synthesis during the development process ( Figure 5). The lignin content and CCR gene expression were gradually increased in tobacco during maturation [48]. Similarly, the transcripts of CsCCR were raised during the stem development of the tea plant, and they showed higher levels in "Fudingdabai" compared to "Suchazao", with the results of higher lignin accumulation in "Fudingdabai" compared to "Suchazao" [49]. The transcription of lignin-synthesis-related genes can be regulated by transcript factors such as MYBs [50,51]. However, in the expression of MsCCR5-7 (Group Ib) and some MsCCR-likes (Group III and MsCCR-like17), a few changes even decreased during the 28 days of plant development ( Figure 5). This may be because of the different cis-element in the promoter of these genes compared to that in Group Ia, II, and IV ( Figure 4). Interestingly, some researchers argue that not all members of the CCR families are involved in lignin synthesis after a long period of evolution [52].
Meanwhile, the expression of MsCCR5-7 (Group Ib), MsCCR-like5-7 (Group III), and MsCCR-like8-11, 23 (Group IV) was upregulated with varying degrees after chilling, salinity, drought, heavy metals, and UV-B stresses ( Figure 6). Several reports have shown that CCR participated in plants coping with environmental stresses. The early response genes to salt stress in the root of melon seedlings include CCR and transcript factor MYB [53]. CCR11 was upregulated under salt and water-deficient conditions in Populus trichocarpa [54]. In addition, the expression patterns of MsCCR1-4 and MsCCR5-7 were different during development and stress response (Figures 5 and 6). This is consistent with a further report that CCR1 is involved in lignification during the development process, while CCR2 is involved in stress responses in Arabidopsis [28]. In addition, the loss function of MtCCR1 showed deficiency in plant growth, but the mutation of MtCCR2 does not exhibit inhibited plant growth [34,42]. The function of MsCCR5-7 (homologous to MtCCR2 in [34]) and most MsCCR-likes might be plant defense against biotic and abiotic stresses by regulating lignin biosynthesis [55,56]. These diverse function and expression profiles of CCRs might be related to their duplication during evolution [57].
The expression of MsCCRs and MsCCR-likes (in particular) exhibited different induced levels under phytohormone SA, ABA, SNP, and ETH treatments ( Figure 6). These findings indicated that CCR participated in phytohormone-mediated development, biotic stress, and abiotic stress responses. For example, the ethylene response factor AP2/ERF Ii049 mediated methyl jasmonate, SA, and ABA response by regulating CCR-associated lignin synthesis [58]. In addition, the expression levels of CCRs after ABA treatment are in accordance with the results of the ABRE element in the promoter of CCRs in alfalfa (Figures 4 and 6). The underlying pathway might include some transcript factors such as MYBs and NACs [23,56,59]. Furthermore, it was found that ETH-treated strawberry showed higher hemicelluloses, cellulose, and neutral sugars than the control [60]. Alinduced pectin and hemicellulose accumulation can be alleviated by SNP treatment in rice root cells [61]. Although the effects of lignin content on stress resistance and feed quality are contradictory, these results suggested the possible role of other composites in the cell walls to solve this contradiction, in other words, with reduced lignin and enhanced resistance.
To further illustrate the functions of the MsCCRs and MsCCR-likes, we cloned and expressed recombinant MsCCR1 and MsCCR-like1, followed by the biochemical analyses (Table 1; Figure S2). The MsCCR1 showed catalytic ability with the substrates feruloyl-CoA, p-coumaroyl-CoA, and sinapoyl-CoA, which is similar to the findings in EgCCR, AtCCR1, and PhCCR1 [62][63][64]. Meanwhile, the substrates p-coumaroyl-CoA and caffeoyl-CoA can be catalyzed by MsCCR-like1, suggesting that MsCCR-likes also play a role in lignin synthesis. Combined with the promoter analysis and expression profile (Figures 4 and 6), the functions of CCR-like might be linked to plant defense response [65]. Chao et al. [38] point out that H2O2(X)2K205 is a crucial motif that can be used to distinguish between CCRs and CCR-like proteins. In this text, the results from three-dimensional structure models showed the primary difference is the missing of the H212(X)2K215R263 motif and the C-terminal end in MsCCR-like1 protein (Figure 7), which also can be found in the results of protein alignment (Figure 3). Prasad et al. [1] concluded that the ARG51, ASN52, ASP54, and ASN58 in CCRs are critical residues for feruloyl binding. In this study, the motif RNXD(X)3N is found in MsCCR1 (NADP specificity box in Figure 3) but not in MsCCR-like1 (with QQYGEXXX instead). Pan et al. [64] found that residues Ile124, Gly125, Val185, Leu186, Ala220, and Tyr284 formed a Ph-CCR1 binding pocket for the phenolic ring. The MsCCR1 is in possession of I134, G135, V195, L196, A206, and Y275 residues, which consist of the reported CCR proteins, but not in MsCCR-like1 and other MsCCR-likes ( Figures S3 and S4). These results highlight the potential use of the CCRs in alfalfa genetic engineering, as well as pharmacologic decreased CCR gene expression by specific chemicals. On the contrary, environmental-stresses-induced CCR gene expression might be helpful for plant defense but might have a negative effect on forage quality. Therefore, the overall quality should also be considered when the genetic approach was implemented targeting lignin-reduced plants. Furthermore, it will be very interesting to study the function of CCR-likes and to compare the structural and functional differences between CCR and CCR-like in future work.

Plant Growth and Treatments
Sterilized alfalfa seeds (M. sativa L. Victoria, Clover Seed & Turf Co., Beijing, China) were germinated at 25 • C for 1 d, and then uniform seeds were selected and transferred to the illuminating incubator in quarter-strength Hoagland's solution [66]. Five-day-old seedlings were used for common environmental stresses and plant hormone treatments. NaCl was used for salinity stress at 100 mM [67]; PEG6000 was used for mimicking drought stress at 15% (w/v) [68]; AlCl 3 was used at 150 µM [69]; and CdCl 2 was used at 100 µM [66]. UV-B treatment was performed using the methods described by Xie et al. [70], 1 J·m −2 ·h −1 treated for 6 h and recovered for another 42 h. Salicylic acid (SA) was used at 50 mg/L [71]; abscisic acid (ABA) was used at 50 mg/L [72]; nitric oxide donor sodium nitroprusside (SNP) was used at 80 µM [61]; and ethephon (ETH) was used at 500 µM immediately after preparation [60]. All the reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).

MsCCR Identification and Classification
For MsCCRs gene determination, a local protein database was established by using DNATOOLS software with alfalfa (M. sativa L.) genomic data [36,73]. To identify candidates of the CCR family, sequences of 2 CCRs from Arabidopsis thaliana (At1G15950 and At1G80820) and 6 CCRs from Medicago truncatula (MTR_2g104960, MTR_4g006940, MTR_5g029990, MTR_6g406250, MTR_2g028620, and MTR_4g009040) were used for BLASTp and BLASTn (E-value ≤ 0.001; Identities ≥ 80%; obtained from NCBI: www.ncbi.nlm.nih. gov, accessed on 30 April 2021) analysis. When comparing, the single exon similarity of each candidate must be above 85%, and the exon deletion number must be less than or equal to 2. After that, all the candidates were blasted against CDS/mRNA database, and a score greater than 80 was determined as a member of MsCCR gene family. Finally, they were confirmed by sequence alignment against the protein database. Regions of resulting signature motifs were identified by a MEGA-X ClustalW sequence alignment, cutoff rate = 30%. If a candidate contains the CCR signature motif "(KNW)YCYGK", it will be named MsCCR, or it will be named MsCCR-like. The pI and MW of amino acids were calculated using ExPASy Compute pI/MW [74].

Motif Analysis and Gene Structure Visualization
Conversed motifs were found with the widely used online tool Multiple Em for Motif Elicitation (MEME; [75]) [76,77]. Detailed settings are below: motif width is between 6 aa and 50 aa; the maximum motif numbers are set to 20; running under classic model. GSDS online tool [78] was used to visualize exon-intron structure.

Multiple Sequence Alignment and Phylogenic Analysis Evolutionary Analysis
The phylogenetic tree was constructed with the neighbor-joining model, JTT method (bootstrap = 400) by MEGA-X [79], and iTOL [80] for decoration. Similarity analysis was conducted through BioEdit [81]. All the sequences used for the phylogenetic tree and alignment are listed in Table S3.

Cis-regulatory Element Analysis and 3D Structure Prediction
First, 2000 bp upstream was extracted from the initiation codon of each gene to ensure that they contained the promotor region. These sequences were used for cis-regulatory element analysis using PlantCARE [82]. Prediction of the 3D structure was conducted by SWISS-MODEL [83] for modeling (GMQE ≥ 0.8) and PyMOL [84] for the exhibition of detailed structure.

Collinearity Analysis and Chromosome Localization
Collinearity analysis was conducted by TBtools [85] One-step MCScanX, and visualized by TBtools Dual Systeny Plot. Chromosome localization was completed by TBtools Gene Location Visualize from GTF/GFF.

RNA Isolation and RT-qPCR Analysis
For the total RNA isolation, 500 mg of alfalfa tissues (leaf, stem, and root) under different stages or after different treatments was homogenized and further extracted by using the RNAiso Plus kit (Takara, Japan, Dalian). Five micrograms of total RNA was used for reverse transcription with the HiScript III 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China). qPCR was carried out on an Applied Biosystems™ QuantStudio™ 5 (ABI, Los Angeles, CA, USA). The reaction contains 2× ChamQ Universal SYBR qPCR Master Mix (Vazyme, China, Nanjing) 10 µL, primer F/R (10 µM) 0.4 µL, template 200 ng, and ddH 2 O makes up to 20 µL. Relative expression levels were calculated by using the 2 −∆∆CT method with Actin2 used as the internal reference. The primer sequences are listed in Table S4.

Lignin Content and Staining Analysis
The main stem was sliced by hand and soaked in 2% phloroglucin (in 95% ethanol) for 5 to 10 s, then treated with 12% muriatic acid for 5 to 10 s until lignin turns red. The time was extended to 20 to 30 s when treating the whole root.

Cloning, Expression, and Purification of Recombinant MsCCR
The target sequences were cloned from the cDNA template. Plasmid pET-28α was used to construct the expression vector. Constructed vectors were stored in Escherichia coli DH5α. The vector was transferred into E. coli BL21 and cultured in Luria-Bertani (LB) medium 37 • C 210 rpm. The recombinant proteins were induced at 16 • C 150 rpm with 200 µM isopropy-β-D -thiogalactoside (IPTG) in LB medium for at least 5 h or overnight when OD 600 = 0.6-0.8. Induced BL21 was centrifuged and resuspended in 1× PBS. The cells were disrupted on ice by a sonic dismembrator with 100 µM phenylmethylsulfonyl fluoride (PMSF). After centrifuging at 8000 rpm 4 • C for 20 min, the liquid was filtered (0.45 µm filter) in BBI™ Ni-NTA 1 mL (Prepacked Gravity Column), then eluted with imidazole of different concentrations (20 mM, 50 mM, 100 mM, 250 mM, or 500 mM) in LE buffer. The His-tagged MsCCR proteins were eluted with 100 mM imidazole. The His-tagged MsCCR proteins were eluted with 100 mM imidazole. The purification of obtained proteins was valued by Western blot using an anti-His tag antibody.

CCR Activity and Enzyme Kinetic Parameter Analysis
The protein eluted by 100 mM imidazole was dialyzed in 1 × PBS with 1 mM dithiothreitol (DTT), magnetic stirring on ice for 3 h. BCA Protein Assay Kit (Takara, China, Dalian) was used to detect the protein content of dialyzed enzymes. For enzymatic analysis, a 500 µL reaction was used, containing the following content: enzyme 20 µg, NADPH 160 µM, substrate (feruloyl-CoA, p-coumaroyl-CoA, caffeoyl-CoA, and sinapoyl-CoA) (ZZBIO CO., LTD, China, Shanghai) 30-150 µM, 1 × PBS to 500 µL. The decrease of A 366 was recorded, and changed the concentration of different substrates. The parameters K m , V max , and K cat will be calculated by V 0 /[S] − V 0 plots.

Conclusions
The present study identified 30 CCRs in M. sativa genome, and further analyzed the phylogenetics and evolution of CCR family proteins. Seven MsCCR genes were located on seven chromosomes in alfalfa, which are homologous to two CCR genes in M. truncatula. Meanwhile, 23 MsCCR-like genes were categorized into four subfamilies. The expression patterns in alfalfa seedlings at different growth stages suggested that MsCCR genes play an important role in cell wall lignification. In addition, both MsCCR and MsCCR-like genes responded to environmental stresses and hormone treatment, which is consistent with the cis-elements in their promoters. Furthermore, the biochemical and three-dimensional structure model analysis of recombinant MsCCR1 and MsCCR-like1 suggested the importance of some motifs for catalytic activities under the specific substrates.

Conflicts of Interest:
The authors declare no conflict of interest.